Genetic factors that influence moenomycin production in streptomycetes.
ABSTRACT: Moenomycin, a natural phosphoglycolipid product that has a long history of use in animal nutrition, is currently considered an attractive starting point for the development of novel antibiotics. We recently reconstituted the biosynthesis of this natural product in a heterologous host, Streptomyces lividans TK24, but production levels were too low to be useful. We have examined several other streptomycetes strains as hosts and have also explored the overexpression of two pleiotropic regulatory genes, afsS and relA, on moenomycin production. A moenomycin-resistant derivative of S. albus J1074 was found to give the highest titers of moenomycin, and production was improved by overexpressing relA. Partial duplication of the moe cluster 1 in S. ghanaensis also increased average moenomycin production. The results reported here suggest that rational manipulation of global regulators combined with increased moe gene dosage could be a useful technique for improvement of moenomycin biosynthesis.
Project description:Streptomyces ghanaensis ATCC14672 is remarkable for its production of phosphoglycolipid compounds, moenomycins, which serve as a blueprint for the development of a novel class of antibiotics based on inhibition of peptidoglycan glycosyltransferases. Here we employed mariner transposon (Tn) mutagenesis to find new regulatory genes essential for moenomycin production. We generated a library of 3000 mutants which were screened for altered antibiotic activity. Our focus centred on a single mutant, HIM5, which accumulated lower amounts of moenomycin and was impaired in morphogenesis as compared to the parental strain. HIM5 carried the Tn insertion within gene ssfg_01967 for putative tRNA (N6-isopentenyl adenosine(37)-C2)-methylthiotransferase, or MiaB, and led to a reduced level of thiomethylation at position 37 in the anticodon of S. ghanaensis transfer ribonucleic acid (tRNA). It is likely that the mutant phenotype of HIM5 stems from the way in which ssfg_01967::Tn influences translation of the rare leucine codon UUA in several genes for moenomycin production and life cycle progression in S. ghanaensis. This is the first report showing that quantitative changes in tRNA modification status in Streptomyces have physiological consequences.
Project description:Moenomycin A (MmA) is a member of the phosphoglycolipid family of antibiotics, which are the only natural products known to directly target the extracellular peptidoglycan glycosyltransferases involved in bacterial cell wall biosynthesis. The structural and biological uniqueness of MmA make it an attractive starting point for the development of new antibacterial drugs. In order both to elucidate the biosynthesis of this unusual compound and to develop tools to manipulate its structure, we have identified the MmA biosynthetic genes in Streptomyces ghanaensis (ATCC14672). We show via heterologous expression of a subset of moe genes that the economy of the MmA pathway is enabled through the use of sugar-nucleotide and isoprenoid building blocks derived from primary metabolism. The work reported lays the foundation for genetic engineering of MmA biosynthesis to produce novel derivatives.
Project description:The moenomycins are phosphoglycolipid antibiotics produced by Streptomyces ghanaensis and related organisms. The phosphoglycolipids are the only known active site inhibitors of the peptidoglycan glycosyltransferases, an important family of enzymes involved in the biosynthesis of the bacterial cell wall. Although these natural products have exceptionally potent antibiotic activity, pharmacokinetic limitations have precluded their clinical use. We previously identified the moenomycin biosynthetic gene cluster in order to facilitate biosynthetic approaches to new derivatives. Here, we report a comprehensive set of genetic and enzymatic experiments that establish functions for the 17 moenomycin biosynthetic genes involved in the synthesis of moenomycin and variants. These studies reveal the order of assembly of the full molecular scaffold and define a subset of seven genes involved in the synthesis of bioactive analogues. This work will enable both in vitro and fermentation-based reconstitution of phosphoglycolipid scaffolds so that chemoenzymatic approaches to novel analogues can be explored.
Project description:Unlike the majority of actinomycete secondary metabolic pathways, the biosynthesis of peptidoglycan glycosyltransferase inhibitor moenomycin in Streptomyces ghanaensis does not involve any cluster-situated regulators (CSRs). This raises questions about the regulatory signals that initiate and sustain moenomycin production. We now show that three pleiotropic regulatory genes for Streptomyces morphogenesis and antibiotic production-bldA, adpA and absB-exert multi-layered control over moenomycin biosynthesis in native and heterologous producers. The bldA gene for tRNA(Leu)UAA is required for the translation of rare UUA codons within two key moenomycin biosynthetic genes (moe), moeO5 and moeE5. It also indirectly influences moenomycin production by controlling the translation of the UUA-containing adpA and, probably, other as-yet-unknown repressor gene(s). AdpA binds key moe promoters and activates them. Furthermore, AdpA interacts with the bldA promoter, thus impacting translation of bldA-dependent mRNAs-that of adpA and several moe genes. Both adpA expression and moenomycin production are increased in an absB-deficient background, most probably because AbsB normally limits adpA mRNA abundance through ribonucleolytic cleavage. Our work highlights an underappreciated strategy for secondary metabolism regulation, in which the interaction between structural genes and pleiotropic regulators is not mediated by CSRs. This strategy might be relevant for a growing number of CSR-free gene clusters unearthed during actinomycete genome mining.
Project description:The biosynthesis of the phosphoglycolipid antibiotic moenomycin A attracts the attention of researchers hoping to develop new moenomycin-based antibiotics against multidrug resistant Gram-positive infections. There is detailed understanding of most steps of this biosynthetic pathway in Streptomyces ghanaensis (ATCC14672), except for the ultimate stage, where a single pentasaccharide intermediate is converted into a set of unusually modified final products. Here we report that only one gene, moeH5, encoding a homologue of the glutamine amidotransferase (GAT) enzyme superfamily, is responsible for the observed diversity of terminally decorated moenomycins. Genetic and biochemical evidence support the idea that MoeH5 is a novel member of the GAT superfamily, whose homologues are involved in the synthesis of various secondary metabolites as well as K and O antigens of bacterial lipopolysaccharide. Our results provide insights into the mechanism of MoeH5 and its counterparts, and give us a new tool for the diversification of phosphoglycolipid antibiotics.
Project description:Here we describe our efforts to improve the levels of phosphoglycolipid antibiotic nosokomycin A production by Streptomyces ghanaensis ATCC14672 via genome engineering approaches. Introduction of two extra copies of leucyl tRNA (UUA) gene bldA and one copy of moenomycin biosynthesis gene cluster led, on average, to threefold increase in nosokomycin A titers (up to 1.5 mg/L). Our results validate genome engineering approach as a viable strategy to improve moenomycin production.
Project description:Cyclic dimeric 3'-5' guanosine monophosphate, c-di-GMP, is a ubiquitous second messenger controlling diverse cellular processes in bacteria. In streptomycetes, c-di-GMP plays a crucial role in a complex morphological differentiation by modulating an activity of the pleiotropic regulator BldD. Here we report that c-di-GMP plays a key role in regulating secondary metabolite production in streptomycetes by altering the expression levels of bldD. Deletion of cdgB encoding a diguanylate cyclase in Streptomycesghanaensis reduced c-di-GMP levels and the production of the peptidoglycan glycosyltransferase inhibitor moenomycin A. In contrast to the cdgB mutant, inactivation of rmdB, encoding a phosphodiesterase for the c-di-GMP hydrolysis, positively correlated with the c-di-GMP and moenomycin A accumulation. Deletion of bldD adversely affected the synthesis of secondary metabolites in S. ghanaensis, including the production of moenomycin A. The bldD-deficient phenotype is partly mediated by an increase in expression of the pleiotropic regulatory gene wblA. Genetic and biochemical analyses demonstrate that a complex of c-di-GMP and BldD effectively represses transcription of wblA, thus preventing sporogenesis and sustaining antibiotic synthesis. These results show that manipulation of the expression of genes controlling c-di-GMP pool has the potential to improve antibiotic production as well as activate the expression of silent gene clusters.
Project description:Transcriptional response of Bacillus subtilis to moenomycin in wild-type 168. Bacillus subtilis 168, WT (-MOE) vs. WT (+MOE). The experiment was conducted in triplicate using three independent total RNA preparations. Untreated samples were labeled with Alexa Fluor 555 and moenomycin treated samples were labeled with Alexa Fluor 647.
Project description:Diguanylate cyclases (DGCs) and phosphodiesterases (PDEs) are essential enzymes deputed to maintain the intracellular homeostasis of the second messenger cyclic dimeric (3'?5') GMP (c-di-GMP). Recently, c-di-GMP has emerged as a crucial molecule for the streptomycetes life cycle, governing both morphogenesis and secondary metabolite production. Indeed, in <i>Streptomyces ghanaensis</i> ATCC14672 c-di-GMP was shown to be involved in the regulatory cascade of the peptidoglycan glycosytransferases inhibitor moenomycin A (MmA) biosynthesis. Here, we report the role of four c-di-GMP-metabolizing enzymes on MmA biosynthesis as well as morphological progression in <i>S. ghanaensis</i>. Functional characterization revealed that RmdA<sub>gh</sub> and CdgA<sub>gh</sub> are two active PDEs, while CdgE<sub>gh</sub> is a DGC. In vivo, overexpression of <i>rmdA<sub>gh</sub></i> and <i>cdgA<sub>gh</sub></i> led to precocious sporulation, whereas overexpression of <i>cdgE<sub>gh</sub></i> and <i>cdgD<sub>gh</sub></i> (encoding a predicted DGC) caused an arrest of morphological development. Furthermore, we demonstrated that individual deletion of <i>rmdA<sub>gh</sub></i>, <i>cdgA<sub>gh</sub>,</i> and <i>cdgD<sub>gh</sub></i> enhances MmA accumulation, whereas deletion of <i>cdgE<sub>gh</sub></i> has no impact on antibiotic production. Conversely, an individual deletion of each studied gene does not affect morphogenesis. Altogether, our results show that manipulation of c-di-GMP-metabolizing enzymes represent a useful approach to improving MmA production titers in <i>S. ghanaensis</i>.
Project description:Streptomyces ghanaensis produces the antibiotic moenomycin A, which is the only known direct inhibitor of bacterial peptidoglycan glycosyltransferases (transglycosylases). Recent progress in understanding moenomycin biosynthesis opens the door to the generation of novel moenomycins via biocombinatorial approaches. To realize the promise of such an approach, one needs better knowledge of the S. ghanaensis genome and diverse genetic tools for stable expression of recombinant constructs in this strain. In this respect, we report the intergeneric Escherichia coli-S. ghanaensis conjugal transfer of plasmids pRT801 and pSOK804 based on the actinophage BT1 and VWB integrase systems, respectively. The attB sites for these two plasmids and for pSET152 were characterized. In particular, sequencing revealed that a putative Arg-tRNA gene serves as an integration site for both phage VWB and pSAM2-like actinomycete integrative and conjugative element recently suggested to be widespread and functional in actinomycetes. The stability of the studied plasmids and their neutrality with respect to antibiotic production warrant their use for manipulations of S. ghanaensis genome.